For certain aspects of biology, three-dimensional (3D) imaging of large samples is necessary, such as, for example, in examining the organization of tissue, in mapping gene expression within a tissue structure, in looking at network interactions, and in studying interconnectivity. Advanced optical microscopy techniques offer unique opportunities to investigate these biological structures and processes in vivo. The ability to image tissues or organisms in three dimensions (3D) and/or over time (4D imaging) permits a wide range of applications in neuroscience, immunology, cancer research, and developmental biology. (See, e.g., Mertz, Curr. Opin. Neurobiol. 14, 610-616, (2004); Kerr, J. N. D. & Denk, W., Nature Reviews Neuroscience 9, 195-205, (2008); Friedl, P., Current Opinion in Immunology 16, 389-393, (2004); Bousso, P., Current Opinion in Immunology 16, 400-405, (2004); Provenzano, P. P., et al., Trends in Cell Biology 19, 638-648, (2009); Keller, P. J., et al., Science 322, 1065-1069 (2008); McMahon, A., et al., Science 322, 1546-1550 (2008); and Mavrakis, M., et al., Development 137, 373-387, (2010), the disclosures of each of which are incorporated herein by reference.) Fundamental light-matter interactions, such as light scattering, absorption, and photo-induced biological toxicity, set the limits on the performance parameters of various imaging technologies in terms of spatial resolution, acquisition speed, and depth penetration (how deep into a sample useful information can be collected). Often, maximizing performance in any one of these parameters necessarily means degrading performance in the others. (See, e.g., Ji, N., et al., Curr. Opin. Neurobiol. 18, 605-616, (2008) and Vermot, J., et al., HFSP Journal2, 143-155 (2008), the disclosures of each of which are incorporated herein by reference.)
An example of the limitations of the current technique can be seen in conventional light sheet (LISH) or Orthogonal Light Sheet (OLM) microscopy. LISH/OLM microscopy is a century-old technology that has seen much development and refinement in recent years, under names ranging from Thin Laser light Sheet Microscopy (TLSM), Selective Plane Illumination Microscopy (SPIM) (FIG. 1A, high-speed imaging of live zebrafish heart), Objective Coupled Planar Illumination (OCPI) (FIG. 1B, high-speed calcium imaging of neurons), ultramicroscopy (FIG. 1C, blood vessel system of mouse embryo), and Digital Scanned Laser Light Sheet Fluorescence Microscopy (DSLM) (FIG. 1D, in toto imaging of developing zebrafish embryo), among others. (See, e.g., Siedentopf, H. & Zsigmondy, R., Ann. Phys.-Berlin 10, 1-39 (1902); Voie, A. H., et al., J. Microsc.-Oxf. 170, 229-236 (1993); Fuchs, E., et al., Opt. Express 10, 145-154 (2002); Huisken, J., et al., Science 305, 1007-1009 (2004); Holekamp, T. F., et al., Neuron 57, 661-672 (2008); Dodt, H. U. et al., Nat. Methods 4, 331-336 (2007); Huisken, J. & Stainier, D. Y. R., Development 136, 1963-1975 (2009); and Keller, P. J. & Stelzer, E. H. K., Curr. Opin. Neurobiol. 18, 624-632 (2008), the disclosures of each of which are incorporated herein by reference.)
In LISH or OLM, as it will be referred to herein, (FIG. 1E) a planar sheet of light is used to illuminate the sample, generating fluorescence signal over a thin optical section of the sample, which is then imaged from the direction orthogonal to the light sheet with a wide-field imaging camera. Axial sectioning results from the thinness of the light sheet, while lateral resolution is determined by the detection optics. The orthogonal geometry between the illumination and detection pathways of OLM, compared to the collinear geometry of conventional microscopes, not only enables higher imaging speed due to the parallel image collection (millions of voxels can be imaged simultaneously), but also reduces phototoxicity since only a single focal plane of the sample is illuminated at a time. The depth penetration of OLM into scattering biological tissue, however, is limited (only slightly better than CLSM), due to (i) the imaging requirement of the wide-field detection that requires ballistic fluorescence photons only and scattered photons would degrade the image quality, and (ii) the light sheet is spatially degraded beyond its original thinness due to scattering, as it is focused deep inside an optically heterogeneous sample.
Although OLM offers a great deal of promise, the need for orthogonal illumination means that all fluorescence imaging is done with scattered photons or photons from the fluorophore. Most of the excitation photons do not enter the imaging objective, nor does OLM use the information inherent in the transmitted or absorbed photons. Ideally, an imaging microscope would use the full measure of excitation photons being input into the system, thereby allowing access to the widest possible selection of samples and obtaining the largest amount of structural data. However, currently there are no microscopes or other imaging devices that can do both OLM microscopy and utilize the data generated by the non-orthogonal excitation photons.
Accordingly, it would be advantageous to develop an optical microscope that allows for the simultaneous use of both orthogonally scattered photons or fluorescence, and the in-plane absorbed or transmitted photons to provide new combinations of imaging capabilities heretofore unobtainable with conventional OLM microscopy techniques.